This application will examine the formation of nicotinic synapses on neurons. Despite much information about the vertebrate neuromuscular junction and a variety of synapses in the CNS, almost nothing is known about postsynaptic components at nicotinic synapses on neurons, other than the identify of the nicotinic acetylcholine receptor (nAChR) itself. Given the prevalence of nicotinic signaling and its implication in a variety of neural functions and disorders, it is becoming increasingly important to understand the assembly and regulation of the molecular structures involved. Nicotinic receptors containing alpha7 subunits (a7-nAChRs) are among the most abundant in the nervous system and regulate calcium-dependent events, due in part to their having a high relative calcium permeability. Recent studies on ciliary ganglion neurons show the receptors are concentrated on somatic spines where they utilize calcium influx to control events ranging from immediate receptor modulation to long-term transcriptional control. Preliminary results suggest that both cadherin-related neuronal receptors (CNRs) and PDZ-containing proteins are associated with nicotinic synapses on the neurons and may play important organizational and regulatory roles. The receptors also form clusters on hippocampal neurons in culture but are often associated with presumptive non-nicotimc synapses where they are regulated by neural activity. These findings provide the basis for the following aims: (1) Complete 3D tomographic EM analysis of a complete nicotinic calyx synapse to elucidate structure-function relationships, (2) Identify molecular components driving nicotinic synapse formation on neurons, beginning with CNRs and PDZ-containing proteins, (3) Examine postsynaptic determinants of a7-nAChR function that control their contributions to synaptic signaling, and (4) Use rat hippocampal cultures to examine the regulation and significance of synaptic a7-nAChRs on mammalian CNS neurons. The experimental approaches include confocal and EM imaging, gene cloning and manipulation of dominant negative constructs, immunocytochemistry and immunopurification, and patch clamp recording in culture and in situ. The expected results should identify postsynaptic components that link nAChRs to regulatory and signal transduction elements and tether them to the cytoskeleton. This information will provide insight into the organization of nicotinic synapses and suggest mechanisms controlling their development and function. The biomedical relevance stems in part from the role of nicotinic signaling in fundamental brain function such as learning and memory, and in related disorders such as Alzheimers disease and nicotine addiction.
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